U.S. patent number 6,482,652 [Application Number 09/814,345] was granted by the patent office on 2002-11-19 for biological particle sorter.
This patent grant is currently assigned to The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Eileen Furlong, David Profitt, Matthew Scott.
United States Patent |
6,482,652 |
Furlong , et al. |
November 19, 2002 |
Biological particle sorter
Abstract
An automated particle sorter having a fluid flow path, which
places single biological particles in an optical cuvette. An
exciting light irradiation system having a light source emits a
source of light through the cuvette. The light excites a
fluorescent substance present on the particle, and the emitted
light is detected by a light detection apparatus containing at
least two detection elements for measuring the fluorescence emitted
from the fluorescent substance. A light separation element
separates the fluorescence from the exciting light. A data
processor compares the signal received from the fluorescent light;
and from the background autofluorescent signal, and according to
pre-set parameters, controls the position of a collection conduit
between two set points. The first being a collection set point for
the collection of objects having a first phenotype and the second
being a set point for the collection of objects having a second
phenotype.
Inventors: |
Furlong; Eileen (Mountain View,
CA), Profitt; David (Los Altos, CA), Scott; Matthew
(Stanford, CA) |
Assignee: |
The Board of Trustees of the Leland
Stanford Junior University (Palo Alto, CA)
|
Family
ID: |
22706549 |
Appl.
No.: |
09/814,345 |
Filed: |
March 21, 2001 |
Current U.S.
Class: |
436/63; 209/3.1;
209/552; 209/576; 209/577; 209/906; 356/436; 356/441; 356/442;
422/73; 422/82.08; 436/164; 436/172 |
Current CPC
Class: |
B07C
5/3425 (20130101); G01N 15/1456 (20130101); G01N
21/645 (20130101); G01N 33/5005 (20130101); G01N
33/582 (20130101); G01N 33/6803 (20130101); G01N
2015/149 (20130101); Y10S 209/906 (20130101) |
Current International
Class: |
B07C
5/342 (20060101); G01N 15/14 (20060101); G01N
21/64 (20060101); G01N 33/58 (20060101); G01N
33/68 (20060101); G01N 33/50 (20060101); G01N
033/48 (); G01N 021/76 () |
Field of
Search: |
;436/63,164,172,72
;422/73 ;209/3.1,552,576,577,579,587,588,906
;356/432,436,440,441,442 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
WO 97/49925 |
|
Dec 1997 |
|
WO |
|
WO 00/11449 |
|
Mar 2000 |
|
WO |
|
Other References
Furlong et al. (Feb. 2001), "Automated Sorting of Live Transgenic
Embryos," Nature Biotechnology, vol. 19:153-156. .
Krasnow et al. (Jan. 4, 1998), "Whole Animal Cell Sorting of
Drosophila Embryos." Science, vol. 251:81-85..
|
Primary Examiner: Wallenhorst; Maureen M.
Attorney, Agent or Firm: Sherwood; Pamela J. Keddie; James
S. Bozicevic, Field & Francis LLP
Government Interests
GOVERNMENT SUPPORT
This invention was made with Government support under contract
N00014-98-10689 awarded by the Navy ONR. The Government has certain
rights in this invention
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent
Application No. 60/191,693, filed Mar. 23, 2000.
Claims
What is claimed is:
1. An automated particle sorter, comprising: a fluid flow path for
passage of biological particles, which comprises an optical
cuvette; a fluid ring over an end of said optical cuvette, wherein
said fluid ring injects fluid into a particle stream as it exits
said optical cuvette; a switch that alters the position of a
collection conduit between two set points for sorting of particles
according to their level of fluorescence; a light irradiation
system comprising a light source that emits light through said
cuvette at a wavelength that causes fluorescence excitement; a
light detection apparatus comprising at least two detection
elements for measuring emitted fluorescence; and a data processor
that receives signals from said light detection elements and
according to pre-set parameters, controls said switch to physically
sort said particles.
2. The particle sorter of claim 1, wherein said light irradiation
system comprises a laser, a filter and focusing optics.
3. The particle sorter of claim 1, wherein said light detection
apparatus comprises one or more light separation elements.
4. The particle sorter of claim 3, wherein said light separation
elements are diachroic mirrors that reflect fluorescent light at a
wavelength below said wavelength that causes fluorescence
excitement.
5. The particle sorter of claim 4, wherein said light detection
elements comprise two or more photomultiplier tubes.
6. The particle sorter of claim 5, wherein said switch is a switch
that moves a collection conduit.
7. The particle sorter of claim 1, wherein said collection conduit
comprises two tubes separated by a thin membrane.
8. The particle sorter of claim 1, wherein said fluid flow path is
an elongated member of square or rectangular cross-sectional
geometry, wherein the positions of said particles in the particles
are constrained.
9. An automated particle sorter, comprising: a fluid flow path for
passage of biological particles, which comprises an optical cuvette
and a high density particle chamber and a low density particle
chamber, wherein the high density particle chamber and low density
particle chambers are connected by a fluid valve controlled by a
data processor, and particle density in said fluid flow path is
maintained at a predetermined level by opening and closing of said
fluid valve; a switch that alters the position of a collection
conduit between two set points for sorting of particles according
to their level of fluorescence; a light irradiation system
comprising a light source that emits light through said cuvette at
a wavelength that causes fluorescence excitement; a light detection
apparatus comprising at least two detection elements for measuring
emitted fluorescence; and a data processor that receives signals
from said light detection elements and according to pre-set
parameters, controls said switch to physically sort said
particles.
10. The particle sorter of claim 9, wherein said low density
particle chamber comprises a magnetic stir bar set on a pin.
11. A method of sorting biological particles according to their
level of fluorescence, the method comprising: suspending said
particles in solution; moving said suspension through a liquid flow
path comprising an optical cuvette through which a light is emitted
at a wavelength that excites a fluorescent compound on the
particles; detecting the level of fluorescence on said particles by
at least two light detection elements, wherein a signal from said
light detection elements is received by a data processor that
controls a switch that alters the position of a collection conduit
between two set points; merging said suspension with a high
velocity fluid flow as said suspension exits said optical cuvette
that forces the particles into said collection conduit; and moving
said collection conduit between said two set points in accordance
with the level of fluorescence associated with said particles.
12. The method of claim 11, wherein said particles are whole
animals of from 10 to 105 cells in size.
13. The method of claim 12, wherein said whole animals are viable
embryos.
14. The method of claim 12, wherein said fluorescent compound is a
protein expressed by and associated with said animal.
15. The method of claim 12, wherein said animals are Drosophila
embryos expressing green fluorescent protein.
Description
INTRODUCTION
In many areas of research, the ability to separate animals or other
large biological particles according to their phenotype is
desirable. For example, there are now thousands of mutant
Drosophila strains available. In fact, a project is underway to
isolate a mutation in every Drosophila gene. However, in a breeding
population three-quarters of the animals carry at least one normal
chromosome and only one-quarter carry two mutant chromosomes. The
ability to separate populations of mutant embryos from their normal
siblings would greatly enhance the molecular and biochemical
studies of these newly identified and uncharacterized genes.
Present technology in cell sorting is limited to the isolation of
individual cells. Through the use of a flow cytometer, cells are
sorted on the basis of their levels of fluorescence. The cells are
placed in a laminar stream of liquid and flowed through a small
opening where a jet in air is formed. When this jet is mechanically
vibrated, it breaks into regularly spaced drops, such that there is
approximately one cell per drop. A cell is then sorted or isolated
by putting an electric charge on the droplet of water, which can
then be deflected according to the charge. However, large
biological particles, such as embryos or small animals, are heavy
and difficult to deflect accurately by such a method. The present
invention addresses this problem by eliminating the need for
deflection.
Relevant Literature and Prior Art
Flow cytometers for use in sorting single cells are described in a
number of publications. Exemplary is U.S. Pat. No. 5,880,474,
issued Mar. 9, 1999, and the references cited therein. Krasnow et
al. (1991) Science 251:81-85 describes the use of a conventional
cell sorter to analyze the cells of a dissociated Drosophila
embryo.
Other particle sorters have been described in the art. For example,
Satake et al., U.S. Pat. No. 5,713,473, issued Feb. 3, 1998
describes a conveyer belt type sorter for beans.
Other art in this field is evident in the COPAS.TM. fluorescence
based sorter (manufactured and sold by Union Biometrica,
Somerville, Mass.). Unlike the present invention, the COPAS sorter
uses a fluid switch to interrupt and redirect particle flow. See
International Patent application WO 00/11449.
SUMMARY OF THE INVENTION
An automated particle sorter is provided, which allows the
separation of large multicellular biological particles, including
embryos, small organisms and the like. The particle sorter provides
a means of sorting multicellular aggregates that are too large to
be sorted with an electrostatic deflection flow cytometer. The
particle sorter comprises a fluid flow path, which places single
biological particles in an optical cuvette. An exciting light
irradiation system directs a source of light through the cuvette.
The light excites a fluorescent substance present on the particle,
and the emitted light is detected by a light detection apparatus
comprising at least two detection elements for measuring the
fluorescence emitted from the fluorescent substance. A light
separation element, such as a dichroic mirror, is employed to
separate the fluorescent light from the exciting light. A data
processor compares the signal received from the fluorescent light;
and from the background autofluorescent signal, and according to
pre-set parameters, controls a mechanical switch that alters the
position of a collection conduit between two set points. The
conduit is composed of at least two tubes separated by a very thin
central wall, e.g. a membrane. Sorting is achieved by moving the
appropriate tube under the fluid stream. The tubes can, in turn
lead to other collection vessels.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram of the particle sorter.
FIG. 2 is an expanded view of the light detection apparatus.
FIG. 3A is an expanded view of the switching apparatus, and FIG. 3B
illustrates the collection conduit.
FIG. 4 is an expanded view of the optical chamber.
FIG. 5 is an expanded view of a liquid flow path.
DETAILED DESCRIPTION OF THE EMBODIMENTS
The particle sorter of the invention provides a means of sorting
multicellular aggregates, such as small animals and embryos, that
are too large to be sorted with a conventional, electrostatic
deflection, flow cytometer. The particles are suspended in a
solution, which is pumped through a narrow flow path in which the
particles are dispersed so as to isolate single particles along the
path. The flow path enters an optical cuvette, through which an
exciting light is emitted. The light passes through the cuvette,
and if a fluorescent substance is present in the cuvette, it will
emit fluorescence. One or more partial reflection mirror such as
dichroic mirrors are employed to separate the fluorescent light
from the exciting light. A light detection system comprising one or
more light detecting elements, e.g. photodiodes, photomultiplier
tubes, etc., receives the light and transmits the information to a
data processor. The data processor controls a switching mechanism
that alters the position of a collection conduit between two set
points. The first is a collection set point for the collection of
desired, or saved, objects and the second is a set point for the
collection of waste. The conduit is composed of at least two tubes
separated by a very thin central wall, e.g. a membrane separation.
Sorting is achieved by moving the appropriate tube under the fluid
stream.
An object of the invention is to provide a means of automated
separation of a sub-population of embryos of a given phenotype from
a larger population of embryos. The sorter can automatically
separate whole embryos that express a fluorescent protein from
those that do not. The invention allows the isolation of large
quantities of genotyped embryos and will facilitate the ability to
study genes and their mutations at a whole genome level.
The invention finds particular use in the sorting of biological
particles that are too large to be sorted by conventional flow
cytometry. Such particles are typically greater than the size of a
single cell, and may be as large as an embryo. For example, a
Drosophila embryos is about 1 mm in length and about 0.1-0.2 mm in
diameter. Such large particles will usually comprise at least about
10 cells, more usually at least about 10.sup.2 cells, frequently as
many as 10.sup.3 cells, and may comprise greater than 10.sup.4
cells.
One may use the sorter in drug testing, to determine the reversion
of mutant phenotypes, including embryonic phenotypes, using
pharmacological agents. The sorted particles can be used for the
isolation of genetic material, including mRNA and DNA, particularly
for the synthesis of cDNA, which is then utilized in the
construction of libraries, as probes, for the synthesis of
microarrays, etc. Proteins can be isolated from the sorted
populations to determine multi-protein complex stability, protein
processing and subcellular localization.
In one embodiment of the invention, the sorter is used in the
separation of Drosophila embryos. In order to maintain mutations in
Drosophila, the chromosome carrying the mutation is in trans to a
special chromosome termed the "balancer chromosome" which ensures
that the mutation is inherited in the next generation. As a
consequence of this only 25% of the embryos produced from these
adults will contain the homozygous mutation. Using current
technology, in order to conduct molecular and pharmaceutical
experiments, tens of thousand of homozygous mutant embryos would
have to be hand sorted in order to separate them from the 75% of
the population that contain the balancer chromosome. Due to the
time consuming and laborious task of hand sorting embryos, the
level of experimentation has been severely restricted.
Balancer fly strains are available that carry a gene encoding a
green fluorescent protein (GFP), and therefore it is possible to
recognize in living embryos the population of homozygous mutants,
as they will not contain GFP.
The animals can also be engineered to contain other detectable
markers. For example many fluorescent proteins such a A. Victoria
green fluorescent protein and derivatives thereof have been
described in the art. Epitopes not normally found in the animal can
be expressed and stained with a fluorescent marker. Alternatively,
enzymes such as -galactosidase can be expressed and detected by
substrate modification. Commonly used bioluminescent reporters emit
in the blue to yellow-green range (250-560 nm). Currently,
luciferase genes from a wide variety of vastly different species,
particularly the luciferase genes of Photinus pyralis (the common
firefly of North America), Pyrophorus plagiophathalamus (the
Jamaican click beetle), Renilla reniformis (the sea pansy), and
several bacteria (e.g., Photorhabdus luminescens and Vibrio spp),
are used as luminescence reporter genes. Amino acid substitutions
in the active sites of luciferase clones may be exploited to alter
wavelength of emission (Kajiyama et al. (1991) Prot. Eng.
4:691).
The ability to routinely isolate mutant embryos can also be applied
to drug testing. The sorter of the invention is used to dispense a
fixed number of embryos into one or more containers, e.g. tubes,
plates comprising multiple wells, and the like. For example, the
save tube can be exchanged for a narrow tube or funnel to sort the
embryos into wells, where a robotic arm may be utilized for
manipulation of the plate. Different drugs or different
concentrations of the same drug can then be added to each container
or well using a robot pipetter. The sorter therefore allows
pharmaceutical screens in mutant embryos for potential drugs that
can reverse the effects of mutations causing a wide variety of
defects, e.g. tumors, neuron path finding, aging and longevity, and
sterility defects. Further, the sorted embryos can be used as a
source of nucleic acids, e.g. to make probes, cDNA libraries, and
the like.
FIG. 1 illustrates the general features of the particle sorter. The
biological particles 11 are suspended in solution in a particle
chamber 12. The embryos can be kept in suspension using a magnetic
stir bar that is placed on a pin in order to prevent crushing the
embryos. The suspension is pumped through a narrow flow path 20.
The flow path is an elongated member of any suitable
cross-sectional geometry, and a diameter that permits even flow of
the particles while maintaining a separation of individual
particles. Preferred is a square or rectangular cross-section,
although circular, oval, etc. geometries may also find use.
In order to achieve the goal of delivering the particles in a
single file manner, the inside dimensions of the delivery tube must
be such that two particles cannot exist side by side. Tubing with
such small dimensions can cause high resistance to fluid flows,
necessitating either high pressures or excessively slow flow rates.
In one embodiment of the invention, relatively large diameter
tubing is used for the collection and transfer of the particles
then transitioning gradually to a smaller dimension glass detection
tube. This gradual transition allows the-particles to accelerate
before entering the much faster fluid flow of the small glass tube,
thereby reducing the frequency of clogging.
The concentration of particles in the suspension is adjusted to a
predetermined level in order to permit passage of a single
particles through the flow path leading to the optical cuvette.
Particles are initially added to a high-density chamber. A
suspension of the high density particles is pumped into a
low-density chamber, and then moves through the exit tube to the
optical cuvette. If the sorting rate drops below a defined
threshold, the computer sends a signal to a fluid valve, which
opens and closes. This fluid valve then diverts the fluid flow to
the high-density chamber. Fluid will then leave this chamber and
enter into the low-density chamber, resulting in the addition of
particles to the low-density chamber. When the rate of particle
sorting increases to the defined threshold, the computer will send
a signal back to the valve and re-direct the fluid flow to the
low-density particle chamber. This will stop the addition of more
particles.
The particle is pumped through the flow path to an optical cuvette
35. The optical cuvette permits passage of light 32 emitted from a
light irradiation system. The light irradiation system comprises a
light source 30 and a filter 31. When a particle 11 is present in
the cuvette, the light will strike it. If a fluorescent dye or
protein is present it will be excited to emit fluorescence with a
wavelength longer than that of the excitation light. This
fluorescence is collected by the focusing optics 50, shown here as
a series of lenses 51 and 52.
After the particles flow through the flow cell they drip from the
end of the cuvette. In one embodiment of the invention a fluid ring
is placed over the end of the cuvette. For example, a metal ring
can be firmly attached onto the end of the cuvette via screws. The
ring injects fluid from a peristaltic pump into the particle stream
as it exits the glass tube, resulting in a high velocity stream of
fluid exiting its orifice. The drop containing the particle enters
this high speed stream of solution. The collection conduit moves
below this stream to sort the particles, but never touches the end
of the cuvette-fluid ring. Increasing the fluid flow after the
point of detection has two advantages: it allows the embryos to
travel at a much slower rate through the point of detection and it
allows the sorting switch to operates at moderate speeds, greatly
simplifying its design.
The fluorescence is transmitted through a light separation element
60. The light signal is received by the detection system, and the
output signal 71 received by a data processor 80. The data
processor analyzes the information and determines according to
pre-set parameters which collection device to sort the particle
into. The data processor may calculate a plurality of
characteristic parameters, indicating characteristics of each
particle based on the generated signal; distribution preparation
device for preparing a distribution of the characteristic
parameters and displaying the distribution on the display; data
storage device for storing information; decision device for
comparison of signals, etc. Typically the sorter will utilize a
microcomputer comprising a signal processing circuit, a CPU, a ROM
and a RAM. The data processor then controls a mechanical switch 40,
which moves the collection conduit 46 to intercept the flow of
particles. The sorted particles may then be further led to
collection vessels 41 and 42.
FIG. 2 shows a more detailed view of light detection system. The
emitted light 33 is directed to a light separation element 60,
which may include one or more partial reflection mirrors such as
dichroic mirrors, and combination prisms, and may be any article
that transmits a part of fluorescence light and reflects the rest
of the fluorescence light. A transparent plate such as an optical
glass plate and a quartz glass plate are useful therefore when the
intensity of the fluorescence is sufficiently high. Generally,
however, a partial reflection mirror such as a dichroic mirror is
preferred, since it introduces the fluorescence light to the
detection elements with high efficiency. The transparent plate or
the dichroic mirror is placed at an angle of 45.degree. to the
optical axis. The element plate is preferably thinner, since the
thickness causes deviation of the optical axis, and has usually a
thickness of about 1 mm. A combination prism constituted of two
isosceles right triangle prisms glued together at the hypotenuse
faces is suitable for achieving highly precise equivalence because
the prism does not cause deviation of the optical axis of the
transmitted light. Wavelength selectivity can be achieved by
forming a multi-layered film at the glued interface.
Optionally, the first partial mirror is used as detection of a
triggering event and to filter the exciting light. For example,
where the light source emits at 488 nm and the fluorescent
substance emits at 510 nm, then the first partial mirror may
reflect at about 500 nm. The reflected light 63 is detected by any
convenient light detecting element 72. The detection element may
include photodiodes, phototransistors, and photomultipliers, but is
not limited thereto.
A second light separation element 62 will usually be included. The
second light separation element reflects the fluorescent light at a
wavelength slightly higher than the peak emission wavelength of the
fluorescent substrate. For example, where the fluorescent substrate
is GFP, having a peak at 510 nm, then the light separation element
may pass through light at greater than 520 nm, and reflect shorter
wavelengths. Optionally the fluorescent light may be transmitted
through an absorption filter 65 that transmits only light having a
wavelength longer than a specified wavelength region. The
fluorescence transmitted through the absorption filter is detected
by the light detection element 73. The fluorescent light is
detected, preferably by a photomultiplier, and the signal
transmitted to a data processor 80.
There is generally an autofluorescent or background signal at a
wavelength higher than that emitted from the fluorescent compound.
For example, it has been found that Drosophila embryos have a high
autofluorescent signal. This background light 34 is received by a
light detection element 74, and the transduced signal transmitted
to the data processor.
The light detection apparatus of this embodiment is characterized
by the pair of detection elements 73 and 74, which are preferably
arranged to be optically equivalent to each other relative to the
light-separating element, meaning the arrangement of the two
detection elements equalizes the changes of output signals from the
two detection elements. The ratio of the changes in the detection
elements, and thereby the intensity of the fluorescence is
measured. The ratio determines whether a given particle is
characterized as "positive" or "negative" for the fluorescent
substance. Where there is an increase in the detection of
fluorescence at the wavelength associated with the marker, relative
to the autofluorescent light, then the particle is considered
"positive". The level of signal required is pre-set, and determined
by various factors, including levels of the substance present on
the particle, number of cells or size of particle,
autofluorescence, etc.
FIGS. 3A and 3B illustrate features of the switching mechanism. The
switching mechanism 40 controls the position of a collection
conduit 46 between two set points: the first point being a
collection set point for the collection of objects having a first
phenotype and the second being a set point for the collection of
objects having a second phenotype. The first and second phenotypes
will usually correspond to high and low levels of fluorescence. In
some embodiments of the invention, the first phenotype can
correspond to a desired phenotype, and will be saved, while the
second phenotype will be collected as waste. Alternatively, both
phenotypes can be saved.
A variety of switches can be used in the sorter, including opposing
solenoid valves that alternatively move the switching chamber,
electromagnetic switches, and the like. For example, this assembly
can be suspended between two electromagnets. Applying electric
current to the electromagnets exerts force on the suspended magnet
in one direction, moving the conduit. Reversing the current
produces a force in the opposite direction.
The flow of particles 20 is merged with a high velocity fluid flow
44 which forces the particles to a switching chamber 40, which
comprises a collection conduit 46 that is mechanically moved by a
switch 45, which is controlled by a signal from the data processor
81. The conduit is composed of two tubes for collecting the two
sorted phenotypes, 47 and 48, which separated by a very thin
central wall 49, which may be a membrane or other very thin
material. Sorting is achieved by moving the appropriate tube under
the fluid stream.
FIG. 4 is a detailed view of the optical cuvette 35. The exciting
light source 30 is passed through a filter 31 to emit a beam of
light 32 through a transparent window 13. An aperture is preferably
provided near the light source to limit the optical path. The
window provides a light path through the flow path 20, which is
encase in a sheath 12. The light emitted from the particle is
passed through focusing optics 50. Usually a lens is employed as
the light focusing element. The curvature radius of the lens is
selected suitably depending on the position of the beam. The lens
system is generally constituted of two lens units, where each lens
unit may be a single lens or a combination lens. The lens may be a
double convex lens or a plano-convex lens.
In one embodiment of the invention, the flow path has the structure
as shown in FIG. 5. In some instances there is found to be variable
background light from the particles, which variability can be part
be attributed to the movement of irregularly shaped particles in
the flow. This is addressed by the use of a square or rectangular
geometry for the flow path 20. The diagonal dimensions of the flow
path orient the particles so that they must travel in a restricted
configuration, thereby reducing the variability of signal.
EXPERIMENTAL
The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and
description of how to make and use the subject invention, and are
not intended to limit the scope of what is regarded as the
invention. Efforts have been made to ensure accuracy with respect
to the numbers used (e.g. amounts, temperature, concentrations,
etc.) but some experimental errors and deviations should be allowed
for. Unless otherwise indicated, parts are parts by weight,
molecular weight is average molecular weight, temperature is in
degrees centigrade; and pressure is at or near atmospheric.
It is to be understood that this invention is not limited to the
particular methodology, protocols, cell lines, animal species or
genera, and reagents described, as such may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
As used herein the singular forms "a", "and", and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a cell" includes a plurality of
such cells and reference to "the embryo" includes reference to one
or more embryos and equivalents thereof known to those skilled in
the art, and so forth. All technical and scientific terms used
herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs unless
clearly indicated otherwise.
All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. The
citation of any publication is for its disclosure prior to the
filing date and should not be construed as an admission that the
present invention is not entitled to antedate such publication by
virtue of prior invention.
EXAMPLE 1
Sorting of Drosophila Embryos
Drosophila embryos are placed in an embryo chamber and suspended in
solution by gentle mixing using a magnetic stir plate. The embryo
chamber is a sealed container with one opening at the top where
solution is flowing in at a fixed rate and there is a second exit
hole on the bottom. This creates a continuous flow of liquid
through the chamber into which an embryo randomly enters. Once an
embryo has exited the embryo chamber it enters a small diameter
glass tube. The glass tube is embedded in a plastic sleeve that has
a window for optical viewing.
Light from an argon laser is enters this window at one side of the
tube, and on the other side of the tube is a series of lenses,
diachronic mirrors and filters, and two photomultiplier tubes. Once
an embryo has passed through this tune it is excited by the laser
light and then any light emitted by the embryo is detected by the
photomultipliers. While the machine is currently used for green
fluorescent protein, it can be easily modified for use with other
fluorochromes, such as luciferase, yellow fluorescent protein, blue
fluorescent protein, cyan fluorescent protein, etc.
The S65T form of GFP is excited at 488 nm and emits light at 510
nm. Once the laser excites the GFP in the embryos, the emitted
light is passed through a focusing mirror and then through a 500 nm
long pass diachroic mirror. The light between 488-500 nm is
detected by a diode, and a second diachroic mirror splits the light
above 500 nm. Light between 500-520 nm is detected by a first
photomultiplier, and the light between 520-600 nm is detected by a
second photomultiplier as background fluorescence. A ratio is taken
from the signal received by the two photomultipliers, i.e. the
difference in fluorescence between GFP wavelengths and background
intrinsic autofluorescence. If this ratio is above a defined
threshold, indicating that the embryo contains GFP, a signal is
sent to a mechanical switching device to direct the flow towards
the waste. If the signal from the first photomultiplier is low,
indicating that the embryo does not contain GFP, the switch directs
the fluid flow to save that embryo in a collection container.
The amplitudes of light emitted from the embryos has been
visualized by an oscilliscope, but in a preferred embodiment is
connected to a computer. The computer allows for a highly sensitive
system that can determine differences in fluorescence even when
there is only a small percentage of GFP containing cells in each
embryo. The computer program also allows the user to save the
parameters between runs.
* * * * *